PROJECT SUMMARY Gram-negative Stenotrophomonas maltophilia (Sm) is recently recognized as an important opportunistic and nosocomial pathogen. Pneumonia is the most frequent expression of Sm infection, although Sm is also linked to bloodstream, eye, skin, and urinary-tract infections. Sm is also an independent risk factor for lung exacerbations in CF patients. Despite the emergence of Sm as a pathogen, due in part to its drug resistance, our understanding of Sm virulence is very limited. Sm is considered an extracellular pathogen, capable of attaching to a variety of host cells, and we have recently shown that Sm encodes a type II secretion system which secretes a protease that triggers apoptosis in lung epithelia. After examining the genome database, we determined that Sm likely also encodes a type IV secretion system (T4SS). In other bacteria, T4SS mediate the delivery of DNA and/or protein ?effectors? into eukaryotic and/or bacterial targets. In order to understand the role of Sm T4SS, we made a mutant devoid of activity by eliminating the VirB10 component of the secretion apparatus, and then tested the mutant in various infection assays. The mutant induced a higher level of apoptosis upon infection of human lung epithelial cells, suggesting that a Sm T4SS effector(s) has anti- apoptotic activity. However, when we infected human macrophages, the virB10 mutant triggered a lower level of apoptosis, implying that Sm T4SS also elaborates a pro-apoptotic factor(s). In both cases, the effect of T4SS required Sm contact with the host cell. The importance of Sm T4SS was also evident when we assessed virB10 mutant growth in the lungs of mice. Moreover, T4SS promoted the growth of Sm when the bacterium was co-cultured with E. coli or P. aeruginosa, suggesting that another effector(s) has anti-bacterial activity. Our bioinformatic analysis of the Sm genome revealed ~18 putative T4SS effectors, including a homolog of a eukaryotic peptidase as well as novel proteins that lack similarity to known proteins. Two-hybrid analysis confirmed that these effectors indeed bind to the T4SS apparatus. Thus, we posit that, following Sm attachment to target cell surfaces, the T4SS apparatus delivers multiple (novel) effectors into the cytoplasm, with some of those effectors inducing or modulating death pathways. That a T4SS can have anti- and pro- apoptotic effects on different targets, including both human and bacterial cells, has, to our knowledge, not been seen before. Thus, we propose to identify the effectors that i) mediate the pro- and anti-apoptotic action on human cells (Aim 1), and ii) impact other bacteria as well as amoebal hosts for Sm (Aim 2). Besides giving much-needed understanding of Sm, the data obtained will have implications for the many undefined T4SS that exist in the genomes of other bacteria and aid in our appreciation of T4SS as a target for new anti-microbials.